Pharmaceutical combinations for treating cancer

ABSTRACT

The present invention is directed to a combination comprising a proteasome inhibitor and a compound of formula I or a pharmaceutically acceptable salt thereof: 
     
       
         
         
             
             
         
       
     
     to a pharmaceutical composition and to a kit both comprising said combination, to the combination, composition or kit for use in the treatment of cancer, and to a method of treatment of cancer in a patient in need thereof comprising administering to said patient an effective amount of said combination, composition or kit.

TECHNICAL FIELD

The present invention relates to combinations and compositions that areof use in the treatment of cancer, for example in the treatment ofbreast cancer or of hematologic cancers such as multiple myeloma,lymphoma or leukemia.

BACKGROUND TO THE INVENTION

Cancer is one of the most life threatening diseases. Cancer is acondition in which cells in a part of the body experience out-of-controlgrowth. According to latest data from American Cancer Society, it isestimated there will be 1.67 million new cases of cancer in USA in 2014.Cancer is the second leading cause of death in the United States (secondonly to heart disease) and will claim more than 585,000 lives in 2014.In fact, it is estimated that 50% of all men and 33% of all women livingin the United States will develop some type of cancer in their lifetime.Therefore cancer constitutes a major public health burden and representsa significant cost in the United States. These figures are reflectedelsewhere across most countries globally, although the types of cancerand relative proportions of the population developing the cancers varydepending upon many different factors such including genetics and diet.

For decades surgery, chemotherapy, and radiation were the establishedtreatments for various cancers. Patients usually receive a combinationof these treatments depending upon the type and extent of their disease.But chemotherapy is the most important option for cancer patients whensurgical treatment (i.e. the removal of diseased tissue) is impossible.While surgery is sometimes effective in removing tumors located atcertain sites, for example, in the breast, colon, and skin, it cannot beused in the treatment of tumors located in other areas, such as thebackbone, nor in the treatment of disseminated hematologic cancersinclude cancers of the blood and blood-forming tissues (such as the bonemarrow). They include multiple myeloma, lymphoma and leukemia. Radiationtherapy involves the exposure of living tissue to ionizing radiationcausing death or damage to the exposed cells. Side effects fromradiation therapy may be acute and temporary, while others may beirreversible. Chemotherapy involves the disruption of cell replicationor cell metabolism. It is used most often in the treatment of breast,lung, and testicular cancer. One of the main causes of failure in thistreatment of cancer is the development of drug resistance by the cancercells, a serious problem that may lead to recurrence of disease or evendeath. Thus, more effective cancer treatments are needed. Multiplemyeloma is a significant and growing problem. It is a cancer arisingfrom plasma cells. Normal plasma cells produce immunoglobulins to fightinfection. In myeloma, the plasma cells become abnormal, multiplyuncontrollably and release only one type of antibody—known asparaprotein—which has no useful function. It tends to accumulate in thebone marrow and circulate in the blood and can be detected in the urineas well. It affects multiple sites in the body (hence ‘multiple’myeloma) where bone marrow is normally active in adults. The main formsof multiple myeloma (or myeloma as it is also referred to) are activemyeloma, plasmacytoma, light chain myeloma and non-secretory myeloma.The number of new cases of myeloma in the US in 2011 was 6.1 per 100,000men and women per year and the percentage survival rate beyond fiveyears was 45%. It is estimated that the number of new cases in the US in2014 will be over 24,000 (1.4% of all cancer cases), while the number ofdeaths in 2014 will be just over 11,000 (1.9% of all cancer cases).

In WO-A-2010/085377, the compound of formula I was shown to haveexcellent in vitro activity against multiple myeloma cell lines, withactivities in the range of ×35-100 greater than the activity shown bybendamustin.

Leukemia is a type of cancer of the blood or bone marrow characterizedby an abnormal increase of immature white blood cells called “blasts”.Instead of producing normal, functioning white blood cells to fightinfection the body produces large numbers of these non-functionalblasts. Leukemia is a broad term covering a spectrum of diseases. Inturn, it is part of the even broader group of diseases affecting theblood, bone marrow and lymphoid system, which are all known ashematological neoplasms. The most common forms are acute lymphoblasticleukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloidleukemia (AML) and chronic myeloid leukemia (CML), with less commonforms including hairy cell leukemia (HCL), T-cell prolymphocyticleukemia (T-PLL), large granular lymphocytic leukemia and T-cell acutelymphoblastic leukemia. It is estimated that the number of new cases inthe United States in 2014 will be over 52,000 (3.1% of all new cancersin the US) with over 24,000 deaths (4.1% of all cancer deaths in theUS). The percentage survival rate over five years is currently 57.2%, afigure significantly lower than for many other cancers, with thesurvival rate over five years for acute myeloid leukemia beingparticularly low at only 20%.

Lymphoma is a cancer of the lymphatic system. There are two main typesof lymphoma, namely Hodgkin lymphoma and non Hodgkin lymphoma.

Non Hodgkin lymphoma is the more common form of lymphoma. The lymphaticsystem runs throughout the body, and it is therefore possible to findnon Hodgkin lymphoma in almost all parts of the body. In patients withnon Hodgkin lymphoma, some of their white blood cells (lymphocytes)divide abnormally. They do not have any resting time like normal cellsand they start to divide continuously, so too many are produced. They donot naturally die off as they usually do. These cells start to dividebefore they are fully mature and therefore cannot fight infection asnormal white blood cells do. All the abnormal lymphocytes start tocollect in the lymph nodes or other places such as the bone marrow orspleen. They can then grow into tumours and begin to cause problemswithin the lymphatic system or the organ in which they are growing. Forexample, if a lymphoma starts in the thyroid gland it can affect thenormal production of thyroid hormones. There are many different types ofnon Hodgkin lymphoma. They can be classified in several different ways.One way is by the type of cell affected. In non Hodgkin lymphoma twotypes of lymphocyte can be affected—B cells and T cells. This isclassified as B cell lymphoma or a T cell lymphoma. Most people with nonHodgkin lymphoma have B cell lymphomas. T cell lymphomas are more commonin teenagers and young adults.

The cells of Hodgkin lymphoma have a particular appearance under themicroscope. These cells are called Reed Sternberg cells. Non Hodgkinlymphomas do not have Reed Sternberg cells. It is important for doctorsto be able to tell the difference between Hodgkin lymphoma and nonHodgkin lymphoma cells as they are two different diseases. In Hodgkinlymphoma, it is cells in the lymph nodes that have become cancerous.

The % survival rate over 5 years in 2009 for patients with non Hodgkinlymphoma was 63%, while the survival rate for those with Hodgkinlymphoma over the same period was 83%.

Breast cancer is a cancer that forms in tissues of the breast. The mostcommon type of breast cancer is ductal carcinoma, which begins in thelining of the milk ducts (thin tubes that carry milk from the lobules ofthe breast to the nipple). Another type of breast cancer is lobularcarcinoma, which begins in the lobules (milk glands) of the breast.Breast cancers can be classified into sub-groups as claudin-low tumors,basal-like tumors, human epidermal growth factor receptor 2 (HER2)positive tumors, luminal A tumors and luminal B tumors. Invasive breastcancer is breast cancer that has spread from where it began in thebreast ducts or lobules to surrounding normal tissue. Breast canceroccurs in both men and women, although male breast cancer is rare. In2014, it is estimated that there will be nearly 233,00 new cases inwomen and 2,400 in men, with 40,00 female deaths and just over 400 maledeaths.

Approximately 15 out of every 100 women with breast cancer havetriple-negative breast cancer, i.e. are estrogen negative, areprogesterone negative and are HER2 negative. Recurrent triple-negativebreast cancer is a condition of high unmet medical need, due to itsaggressive biology, fast development of drug resistance and lack ofmolecular targets. Until now, chemotherapy remains the standard of carefor advanced triple-negative breast cancer with a poor median overallsurvival.

In WO-A-2010/085377, the compound of formula I below is disclosed. It isa first-in-class dual-functional alkylating-HDACi fusion molecule whichpotently inhibits the HDAC pathway.

Biological assays showed that the compound of formula I potentlyinhibits HDAC enzyme (HDAC1 IC₅₀ of 9 nM) and it has been shown to haveexcellent in vitro activity against multiple myeloma cell lines.

There is a need for more effective cancer treatments, including thetreatment of breast cancer and of hematologic cancers such as multiplemyeloma, lymphoma or leukemia. Currently, these conditions affect manypeople and the medium to long-term prognosis is not good for many ofthese conditions.

SUMMARY OF THE INVENTION

In a first aspect of the present invention there is provided acombination comprising a proteasome inhibitor and a compound of formulaI or a pharmaceutically acceptable salt thereof:

It has surprisingly been discovered that combinations of a compound offormula I or a pharmaceutically acceptable salt thereof and a proteasomeinhibitor such as carfilzomib or bortezomib are particularly effectivein the treatment of cancers including hematologic cancers such asmultiple myeloma, lymphoma and leukemia, and breast cancer, such thatthey are highly promising in efforts to address the problem of findingmore effective treatments for cancer. The combinations may optionallyfurther comprise a glucocorticoid such as dexamethasone. These furthercombinations are also particularly effective in the treatment of cancer.

In a second aspect of the present invention, there is provided apharmaceutical composition comprising a pharmaceutically acceptablediluent or carrier and a combination according to the first aspect ofthe invention.

In a third aspect of the present invention, there is provided a kitcomprising a combination according to the first aspect of the presentinvention and, optionally, instructions for treating a patient.

In a fourth aspect of the present invention, there is provided acombination, composition or kit according to the first, second or thirdaspect of the present invention for use in the treatment of cancer.

In a fifth aspect of the present invention, there is provided a methodof treating cancer in a patient in need thereof comprising administeringto said patient a combination, composition or kit according to thefirst, second or third aspect of the present invention.

In a sixth aspect of the present invention, there is provided a compoundof formula (I) or a pharmaceutically acceptable salt thereof for use inthe treatment of relapsed/refractory multiple myeloma. In oneembodiment, the compound of formula (I) or the pharmaceuticallyacceptable salt thereof is for use in the treatment ofrelapsed/refractory multiple myeloma in combination with a proteasomeinhibitor and optionally further in combination with a glucocorticoid.

In a seventh aspect of the present invention, there is provided a methodof treatment of relapsed/refractory multiple myeloma in a patient inneed thereof comprising administering to said patient a compound offormula (I) or the pharmaceutically acceptable salt thereof. In oneembodiment, the compound of formula (I) or the pharmaceuticallyacceptable salt thereof is administered in combination with a proteasomeinhibitor and may further optionally be administered in combination witha glucocorticoid as well.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of the % surviving in vitro MM1S multiple myeloma cellsas a % of control versus concentration for different tested compoundsafter 48 hours incubation, for single compounds and as combinations(double and triple);

FIG. 2 is a plot of the % surviving in vitro MM1S multiple myeloma cellsas a % of control versus concentration for different tested compoundsafter 72 hours incubation, for single compounds and as combinations(double and triple);

FIG. 3 is a plot of tumour growth (mm³) against the number of days ofstudy for different tested compounds for CB17-SCID mice subcutaneouslyinoculated into the right flank with 3×10⁶ MM1S cells, for singlecompounds and as combinations;

FIG. 4 is a plot of the % surviving in vitro RPM18226 multiple myelomacells as a % of control versus concentration for different testedcompounds after 48 hours incubation, for single compounds and ascombinations (double);

FIG. 5 is a plot of the % surviving in vitro 2013-10-16 MTS AMO abzbmultiple myeloma cells as a % of control versus concentration fordifferent tested compounds after 48 hours incubation, for singlecompounds and as combinations (double);

FIG. 6 is a plot of the % surviving in vitro 2014-01-15 MTS Jeko mantlecell lymphoma cells as a % of control versus concentration for differenttested compounds after 48 hours incubation, for single compounds and ascombinations (double);

FIG. 7 is a plot of the % surviving in vitro 2014-01-15 MTS Grantamantle cell lymphoma cells as a % of control versus concentration fordifferent tested compounds after 48 hours incubation, for singlecompounds and as combinations (double);

FIG. 8 is a plot of the % surviving in vitro 2014-02-21 MTS MTSMDA-MB468 basal like breast cancer cells as a % of control versusconcentration for different tested compounds after 48 hours incubation,for single compounds and as combinations (double);

FIG. 9 is a plot of the % surviving in vitro MTS HL-60 promyelocyticleukemia cells as a % of control versus concentration for differenttested compounds after 48 hours incubation, for single compounds and ascombinations (double);

FIG. 10 is a plot of the % surviving in vitro MTS U937 acute myeloidleukemia cells as a % of control versus concentration for differenttested compounds after 48 hours incubation, for single compounds and ascombinations (double);

FIG. 11 is a plot of the % surviving in vitro BJAB (germinal centerline) B cell lymphoma cells as a % of control versus concentration fordifferent tested compounds after 48 hours incubation, for singlecompounds and as combinations (double);

FIG. 12 is a plot of the % surviving in vitro OciLy3 (ABC-type) B celllymphoma cells as a % of control versus concentration for differenttested compounds after 48 hours incubation, for single compounds and ascombinations (double);

FIG. 13 is a plot of the % surviving in vitro TMD8 (ABC-type) B celllymphoma cells as a % of control versus concentration for differenttested compounds after 48 hours incubation, for single compounds and ascombinations (double);

FIG. 14 is a plot of the % surviving in vitro BT-549 triple negativebreast cancer cells as a % of control versus concentration for differenttested compounds after 48 hours incubation, for single compounds and ascombinations (double); and

FIG. 15 is a plot of % surviving fraction of in vitro T98G, U251MG andU87MG glioblastoma cell lines against dose of radiotherapy (Gy) incombination with two different concentrations of the compound of formulaI (EDO-S101) against a control with radiotherapy alone.

DETAILED DESCRIPTION OF THE INVENTION

In the present application, a number of general terms and phrases areused, which should be interpreted as follows.

“Animal” includes humans, non-human mammals (e.g., dogs, cats, rabbits,cattle, horses, sheep, goats, swine, deer, and the like) and non-mammals(e.g., birds, and the like).

“Pharmaceutically acceptable salts” means salts of compounds of thepresent invention which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity. Suchsalts include acid addition salts formed with inorganic acids, or withorganic acids. Pharmaceutically acceptable salts also include baseaddition salts which may be formed when acidic protons present arecapable of reacting with inorganic or organic bases. Generally, suchsalts are, for example, prepared by reacting the free acid or base formsof these compounds with a stoichiometric amount of the appropriate baseor acid in water or in an organic solvent or in a mixture of the two.Generally, non-aqueous media like ether, ethyl acetate, ethanol,isopropanol or acetonitrile are preferred. Examples of the acid additionsalts include mineral acid addition salts such as, for example,hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, sulfamate,nitrate, phosphate, and organic acid addition salts such as, forexample, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate,succinate, tartrate, salicylate, tosylate, lactate,naphthalenesulphonae, malate, mandelate, methanesulfonate andp-toluenesulfonate. Examples of the alkali addition salts includeinorganic salts such as, for example, sodium, potassium, calcium andammonium salts, and organic alkali salts such as, for example,ethylenediamine, ethanolamine, N,N-dialkylenethanolamine,triethanolamine and basic aminoacids salts.

It has surprisingly been discovered that combinations of a compound offormula I or a pharmaceutically acceptable salt thereof and a proteasomeinhibitor such as carfilzomib or bortezomib are particularly effectivein the treatment of cancers including hematologic cancers such asmultiple myeloma, leukemia and lymphoma, and breast cancer such thatthey are highly promising in efforts to address the problem of findingmore effective treatments for cancer. The combinations may optionallyfurther comprise a glucocorticoid such as dexamethasone. These furthercombinations are also particularly effective in the treatment of cancer.

In the combination of the present invention, the pharmaceuticallyacceptable salt of the compound of formula I may preferably be thehydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, sulfamate,nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate,glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate,tartrate, tosylate, salicylate, lactate, naphthalenesulfonate oracetate, and more preferably the acetate.

In the combination of the present invention, the proteasome inhibitormay preferably be selected from the group consisting of bortezomib,carfilzomib, marizomib, delanzomib (CEP-18770), oprozomib (ONX 0912),ixazomib (MLN-9708) and LU-102, or a pharmaceutically acceptable saltthereof. Particularly preferably, the proteasome inhibitor may beselected from bortezomib, carfilzomib and LU-102.

The structures of these proteasome inhibitors are as follows:

The combination of the present invention may further comprise aglucocorticoid. In this embodiment of the combination of the presentinvention, the glucocorticoid may preferably be selected from the groupconsisting of dexamethasone, fluocinolone acetonide and prednisone, andit is most preferably dexamethasone.

In one further preferred combination of the present invention comprisinga compound of formula I or a pharmaceutically acceptable salt thereof, aproteasome inhibitor and optionally a glucocorticoid, said combinationmay further comprise one or more additional pharmaceutically activeagents. Particularly suitable pharmaceutically active agents areanti-tumor agents having a different mode of action to the compound offormula I or a pharmaceutically acceptable salt thereof, the proteasomeinhibitor and the glucocorticoid, e.g. alkylating agents such asnitrosureas, ethylenimines, alkylsulfonates, hydrazines and triazines,and platinum based agents; plant alkaloids, taxanes, vinca alkaloids;anti-tumor antibiotics such as chromomycins, anthracyclines, andmiscellaneous antibiotics such as Mitomycin and Bleomycin;anti-metabolites such as folic acid antagonists, pyrimidine antagonists,purine antagonists and adenosine deaminase inhibitors; topoisomeraseinhibitors such as topoisomerase I inhibitors, topoisomerase IIinhibitors, miscellaneous anti-neoplastics such as ribonucleotidereductase inhibitors, adrenocortical steroid inhibitor, anti-microtubuleagents, and retinoids; protein kinases; heat shock proteins, poly-ADP(adenosine diphosphate)-ribose polymerase (PARP), hypoxia-induciblefactors (HIF), proteasome, Wnt/Hedgehog/Notch signaling proteins,TNF-alpha, matrix metalloproteinase, farnesyl transferase, apoptosispathway, histone deacetylases (HDAC), histone acetyltransferases (HAT),and methyltransferase; hormonal therapies, vascular disrupting agent,gene therapy, RNAi cancer therapy, chemoprotective agents, antibodyconjugate, cancer immunotherapy such as Interleukin-2, cancer vaccinesor monoclonal antibodies; and preferably DNA damaging agents,anti-metabolites, topoisomerase inhibitors, anti-microtubule agents,EGFR inhibitors, HER2 inhibitors, VEGFR2 inhibitors, BRAF inhibitors,Bcr-Abl inhibitors, PDGFR inhibitors, ALK inhibitors, PLK inhibitors,MET inhibitors, epigenetic agents, HSP90 inhibitors, PARP inhibitors,CHK inhibitors, aromatase inhibitor, estrogen receptor antagonist, andantibodies targeting VEGF, HER2, EGFR, CD50, CD20, CD30, CD33, etc.

In one preferred embodiment of the combination of the present invention,the proteasome inhibitor, the compound of formula I or apharmaceutically acceptable salt thereof and, if present, theglucocorticoid are adapted for administration concurrently, sequentiallyor separately. Preferably, the proteasome inhibitor, the compound offormula I or a pharmaceutically acceptable salt thereof and, if present,the glucocorticoid are adapted for administration concurrently.

In one preferred embodiment of the combination of the present invention,the proteasome inhibitor is selected from bortezomib, carfilzomib andLU-102 and the compound of formula I or a pharmaceutically acceptablesalt thereof is

or the acetate salt thereof. In one embodiment of this combination, thecombination may further comprise a glucocorticoid wherein saidglucocorticoid is dexamethasone.

In one preferred embodiment of the combination of the present invention,the molar ratio of proteasome inhibitor to compound of formula I or apharmaceutically acceptable salt thereof in said combination is from1:1000 to 1000:1. Preferably, the molar ratio of proteasome inhibitor tocompound of formula I or a pharmaceutically acceptable salt thereof insaid combination is from 1:1000 to 10:1, more preferably from 1:800 to1:200 or from 1:5 to 1:0.5, and most preferably it is from 1:700 to1:400 or from 1:3 to 1:0.5, e.g. 1:1000, 1:900, 1:800, 1:700, 1:600,1:500, 1:400, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1 or 1:0.5.

One particularly preferred combination of present invention comprisesthe compound of formula I or the acetate salt thereof and a proteasomeinhibitor selected from bortezomib and carfilzomib, wherein the molarratio of the proteasome inhibitor selected from bortezomib andcarfilzomib to the compound of formula I or a pharmaceuticallyacceptable salt thereof in said combination is from 1:700 to 1:400, e.g.1:700, 1:600, 1:500 or 1:400. Another particularly preferred combinationof the first aspect of the present invention comprises the compound offormula I or the acetate salt thereof and a proteasome inhibitorselected from LU-102, wherein the molar ratio of LU-102 to the compoundof formula I or a pharmaceutically acceptable salt thereof in saidcombination is from 1:3 to 1:0.5, e.g. 1:3, 1:2, 1:1 or 1:0.5.

It has been surprisingly found that combinations comprising a proteasomeinhibitor and a compound of formula I or a pharmaceutically acceptablesalt thereof are synergistic combinations. In other words, the potencyof the combinations was measured with the Calcusyn software (biosoft,Ferguson, Mo., USA), which is based on the Chou Talay method (Chou etal., Adv. Enzyme Regul., 22, 27-55 (1984)), that calculates acombination index (CI) with the following interpretation:

-   -   CI 1>1: antagonist effect, CI=1: additive effect and CI<1        synergistic effect.

It was found in the present work that for many of the dual combinationsof the invention comprising a proteasome inhibitor and a compound offormula I or a pharmaceutically acceptable salt, CI has been found to beless than 1, indicating synergy.

Another preferred embodiment of the combination of the present inventionfurther comprises a glucocorticoid in addition to the proteasomeinhibitor and the compound of formula I or a pharmaceutically acceptablesalt thereof, wherein the molar ratio of proteasome inhibitor to thecompound of formula I or a pharmaceutically acceptable salt thereof tothe glucocorticoid in said combination is from 1:1000:20 to 1000:1:20.Preferably, the molar ratio of proteasome inhibitor to the compound offormula I or a pharmaceutically acceptable salt thereof to theglucocorticoid in said combination is from 1:1000:10 to 1:100:2.Preferably, the molar ratio of proteasome inhibitor to the compound offormula I or a pharmaceutically acceptable salt thereof to theglucocorticoid used in said combination is from 1:1000:5 to 1:200:2,more preferably 1:700:4 to 1:400:3, e.g. 1:1000:5, 1:900:5, 1:800:4,1:700:4, 1:600:4, 1:500:3 or 1:400:3.

One particularly preferred combination of the present inventioncomprises a proteasome inhibitor selected from bortezomib andcarfilzomib, a compound of formula I or the acetate salt thereof anddexamethasone, wherein the molar ratio of the proteasome inhibitorselected from bortezomib and carfilzomib to the compound of formula I orthe acetate salt thereof to dexamethasone in said combination is from1:700:4 to 1:400:3, e.g. 1:700:4, 1:700:3, 1:600:4, 1:600:3, 1:500:3 or1:400:3. Another particularly preferred combination of the first aspectof the present invention comprises a proteasome inhibitor selected fromLU-102, the compound of formula I or the acetate salt thereof anddexamethasone, wherein the molar ratio of LU-102 to the compound offormula I or the acetate salt thereof to dexamethasone in saidcombination is from 1:3:4 to 1:0.5:3, e.g. 1:3:4, 1:3:3, 1:2:4, 1:2:3,1:1:4, 1:1:3 or 1:0.5:3.

It has also been surprisingly discovered that many of the triplecombinations of the present invention comprising a proteasome inhibitor,a compound of formula I or a pharmaceutically acceptable salt thereofand a glucocorticoid are also synergistic combinations, i.e. thecombination index CI has been found to be less than 1.

The pharmaceutical composition according to the second aspect of thepresent invention comprises a pharmaceutically acceptable diluent orcarrier and a combination according to the first aspect of the presentinvention. Preferred compositions of the second invention include thosecomprising the preferred combinations of the present invention asdescribed and exemplified above. The pharmaceutically acceptable diluentor carrier of the pharmaceutical composition according to the secondaspect of the present can be any suitable dispersant, excipient,adjuvant, or other material which acts as a carrier for the activeagents of the combination of the present invention and which does notinterfere with the active agents present in said combination. Examplesof typical pharmaceutically acceptable carriers and diluents may befound in “Remington's Pharmaceutical Sciences” by E. W. Martin and theseinclude water, saline, dextrose solution, serum solution, Ringer'ssolution, polyethylene glycol (e.g PEG400), a surfactant (e.gCremophor), a cyclopolysaccharide (e.g hydroxypropyl-3-cyclodextrin orsulfobutyl ether β-cyclodextrins), a polymer, a liposome, a micelle, ananosphere, etc.

In the third aspect of the present invention, there is provided a kitcomprising a combination according to the first aspect of the presentinvention and, optionally, instructions for treating a patient.Typically, a kit can comprise a compound of formula I orpharmaceutically acceptable salt thereof, a proteasome inhibitor, and aglucocorticoid together with instructions for treating a patient. Eachactive agent can be provided in a suitable container. The kit mayfurther comprise a delivery system, e.g. for the compound of formula Ior pharmaceutically acceptable salt thereof, the proteasome inhibitor orthe glucocorticoid or any combination thereof.

The instructions may advise administering the proteasome inhibitor, thecompound of formula I or a pharmaceutically acceptable salt thereof and,if present, the glucocorticoid of the combination concurrently,sequentially or separately according to variables such as the specificcondition being treated, the state of that condition, the activity ofthe specific compounds employed; the specific combination employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compounds employed; the duration of the treatment; drugs usedin combination or contemporaneously with the specific compoundsemployed; and like factors well known in the medical arts. Preferredkits according to the third aspect of the present invention includethose comprising the preferred combinations of the present invention asdescribed and exemplified above.

In the fourth aspect of the present invention, there is provided thecombination, composition or kit according to the first, second or thirdaspect of the present invention for use in the treatment of cancer.

In the fifth aspect of the present invention, there is provided a methodof treating cancer in a patient in need thereof comprising administeringto said patient the combination, composition or kit according to thefirst, second or third aspect of the present invention.

It has been found that the combinations, compositions and kits of thepresent invention are highly active both in vitro and in vivo against awide variety of tumour cell types. The anti-tumour activity shown bythese double and triple combinations of the present invention, and bythe combinations in the compositions and kits of the present inventionis, in many cases, more than merely additive, showing combinationindexes CI of significantly less than 1, indicating synergy for thesecombinations. This surprising finding is a further support for theparticular effectiveness of the combinations, compositions and kits ofthe present invention in the treatment of cancer.

Examples of cancers which are treatable by the combinations,compositions and kits of the present invention include hematologiccancers such as multiple myeloma, lymphoma and leukemia, breast cancer,lung cancer, colorectal cancer, prostate cancer, testicular cancer,pancreatic cancer, liver cancer, stomach cancer, biliary tract cancer,esophageal cancer, gastrointestinal stromal tumor, cervical cancer,ovarian cancer, uterine cancer, renal cancer, melanoma, basal cellcarcinoma, squamous cell carcinoma, bladder cancer, sarcoma,mesothelioma, thymoma, myelodysplastic syndrome, glioblastoma andmyeloproliferative disease. In particular, the combinations,compositions and kits of the present invention are effective againsthematologic cancer such as multiple myeloma, lymphoma and leukemia, andbreast cancer.

In one embodiment of the combination, composition or kit for use in thetreatment of a cancer according to the fourth aspect of the presentinvention or the method of treatment in accordance with the fifth aspectof the present invention, the cancer is selected from a hematologiccancer and breast cancer.

Where the combination, composition or kit of the present invention isfor use in the treatment of a hematologic cancer, this may preferably beselected from multiple myeloma (e.g. active myeloma, plasmacytoma, lightchain myeloma or non-secretory myeloma, with all forms being treatablein all phases including relapsed and refractory phases), lymphoma (e.g.Hodgkin lymphoma or non-Hodgkin lymphoma) and leukemia [acutelymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acutemyeloid leukemia (AML, including myeloblastic leukemia, acutepromyelocytic leukemia, acute myelomonocytic leukemia, acute monocyticleukemia, acute erythroleukemia and acute megakaryotic leukemia, withall forms being treatable in all phases including relapsed andrefractory phases), chronic myeloid leukemia (CML), hairy cell leukemia(HCL), T-cell prolymphocytic leukemia (T-PLL), large granularlymphocytic leukemia or T-cell acute lymphoblastic leukemia].

Where the combination, composition or kit of the present invention isfor use in the treatment of breast cancer, the breast cancer maytypically be selected from claudin-low tumors, basal-like tumors, humanepidermal growth factor receptor 2 (HER2) positive tumors, luminal Atumors and luminal B tumors, and it is preferably a triple-negativebreast cancer.

In one preferred embodiment of the combination, composition or kit foruse in the treatment of cancer according to the present invention andthe method of treatment of cancer according to the present invention,the proteasome inhibitor, the compound of formula I or apharmaceutically acceptable salt thereof and, if present, theglucocorticoid are administered concurrently, sequentially orseparately. More preferably, the proteasome inhibitor, the compound offormula I or a pharmaceutically acceptable salt thereof and, if present,the glucocorticoid are administered concurrently.

In the combination for use in the treatment of cancer and the method oftreatment of cancer in accordance with the present invention, thecompound of formula I or a pharmaceutically acceptable salt thereof istypically administered to the patient in need thereof at a dosage rangeof 10 to 100 mg/kg body weight patient, and preferably at a dosage rangeof 40 to 80 mg/kg body weight patient. Typically, the proteasomeinhibitor is administered to the patient in need thereof at a dosagerange of 0.01 to 0.3 mg/kg body weight patient, more preferably at adosage range of 0.05 to 0.15 mg/kg body weight patient. Where aglucocorticoid is also administered in the combination, theglucocorticoid is typically administered at a dosage range of from 0.1to 1 mg/kg body weight patient. Preferably, it is administered at adosage range of from 0.3 to 0.5 mg/kg body weight patient.

The therapeutically effective amount of a combination, composition orkit according to the present invention is an amount of the combination,composition or kit which confers a therapeutic effect in accordance withthe fourth and fifth aspects of the present invention on the treatedsubject, at a reasonable benefit/risk ratio applicable to any medicaltreatment. The therapeutic effect may be objective (i.e. measurable bysome test or marker) or subjective (i.e. subject gives an indication ofor feels an effect). An effective amount of the combination, compositionor kit according to the present invention is believed to be one whereinthe compound of formula I or a salt thereof is included in thecombination at a dosage range of from 10 to 100 mg/kg body weightpatient (e.g. 40 to 80 mg/kg body weight such as 40, 50, 60, 70 or 80mg/kg body weight), the proteasome inhibitor is included at a dosagerange of from 0.01 to 0.3 mg/kg body weight patient (e.g. 0.1 to 1 mg/kgsuch as 0.1, 0.2, 0.3, 0.4 or 0.5 mg/kg body weight) and theglucocorticoid is included at a dosage range of from 0.03 to 1 mg/kgbody weight patient (e.g. 0.3 to 0.5 mg/kg body weight patient, such as0.3, 0.4 or 0.5 mg/kg body weight patient).

Effective doses will vary depending on route of administration, as wellas the possibility of co-usage with other active agents. It will beunderstood, however, that the total daily usage of the combinations,compositions and kits of the present invention will be decided by theattending physician within the scope of sound medical judgment. Thespecific therapeutically effective dose level for any particular patientwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or contemporaneously with the specific compound employed;and like factors well known in the medical arts.

The present invention is also directed to the use of a combination,composition or kit according to the first, second or third aspect of thepresent invention in the manufacture of a medicament for the treatmentof cancer, e.g. for the treatment of a hematologic cancer or breastcancer.

Suitable examples of the administration form of the combination,composition or kit of the present invention include without limitationoral, topical, parenteral, sublingual, rectal, vaginal, ocular, andintranasal. Parenteral administration includes subcutaneous injections,intravenous, intramuscular, intrasternal injection or infusiontechniques. Preferably, the combinations, compositions and kits areadministered parenterally. Combinations and compositions of theinvention can be formulated so as to allow a combination or compositionof the present invention to be bioavailable upon administration of thecombination or composition to an animal, preferably human. Compositionscan take the form of one or more dosage units, where for example, atablet can be a single dosage unit, and a container of a combination orcomposition of the present invention in aerosol form can hold aplurality of dosage units.

Preferably the combinations of the present invention are provided in theform of kits. Typically, a kit includes a proteasome inhibitor, acompound of formula I or a pharmaceutically acceptable salt thereof and,optionally, a glucocorticoid. In certain embodiments, a kit can includeone or more delivery systems, e.g. the proteasome inhibitor, thecompound of formula I or a pharmaceutically acceptable salt thereof and,optionally, a glucocorticoid, or any combination thereof, and directionsfor the use of the kit (e.g. instructions for treating a subject). Thesedirections/instructions may advise administering the proteasomeinhibitor, the compound of formula I or a pharmaceutically acceptablesalt thereof and, if present, the glucocorticoid of the combinationconcurrently, sequentially or separately according to variables such asthe specific condition being treated, the state of that condition, theactivity of the specific compounds employed; the specific combinationemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compounds employed; the duration of thetreatment; drugs used in combination or contemporaneously with thespecific compounds employed; and like factors well known in the medicalarts.

The pharmaceutically acceptable diluent or carrier can be particulate,so that the compositions are, for example, in tablet or powder form. Thecarrier(s) can be liquid, with the combinations, compositions or kitsbeing, for example, an oral syrup or injectable liquid. In addition, thecarrier(s) can be gaseous, so as to provide an aerosol compositionuseful in, for example, inhalatory administration. Such pharmaceuticalcarriers can be liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. The carriers can besaline, gum acacia, gelatin, starch paste, talc, keratin, colloidalsilica, urea, and the like. In addition, auxiliary, stabilizing,thickening, lubricating and coloring agents can be used. In oneembodiment, when administered to an animal, the combination, compositionor kit of the present invention and the pharmaceutically acceptablecarriers are sterile. Water is a preferred carrier when the combinationor composition of the present invention is administered intravenously.Saline solutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical carriers also include excipients such as starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The present compositions, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents.

When intended for oral administration, the combination, composition orkit may be in solid or liquid form, where semi-solid, semi-liquid,suspension and gel forms are included within the forms considered hereinas either solid or liquid.

As a solid composition for oral administration, the combination,composition or kit can be formulated into a powder, granule, compressedtablet, pill, capsule, chewing gum, wafer or the like form. Such a solidcomposition typically contains one or more inert diluents, either as asingle tablet comprising all active agents or as a number of separatesolid compositions, each comprising a single active agent of thecombination of the present invention (in the case of the kit). Inaddition, one or more of the following can be present: binders such ascarboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, orgelatin; excipients such as starch, lactose or dextrins, disintegratingagents such as alginic acid, sodium alginate, corn starch and the like;lubricants such as magnesium stearate; glidants such as colloidalsilicon dioxide; sweetening agents such as sucrose or saccharin; aflavoring agent such as peppermint, methyl salicylate or orangeflavoring; and a coloring agent.

When the combination or composition is in the form of a capsule (e.g. agelatin capsule), it can contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol, cyclodextrin or afatty oil.

The combination, composition or kit can be in the form of a liquid, e.g.an elixir, syrup, solution, emulsion or suspension. The liquid can beuseful for oral administration or for delivery by injection. Whenintended for oral administration, a combination, composition or kit cancomprise one or more of a sweetening agent, preservatives, dye/colorantand flavor enhancer. In a combination or composition for administrationby injection, one or more of a surfactant, preservative, wetting agent,dispersing agent, suspending agent, buffer, stabilizer and isotonicagent can also be included. In the kit of the present invention, theliquid components comprising one or more of the active agents of thecomposition may either be combined prior to administration andadministered concurrently or each active agent may be administeredsequentially or separately.

The preferred route of administration is parenteral administrationincluding, but not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural,intranasal, intracerebral, intraventricular, intrathecal, intravaginalor transdermal. The preferred mode of administration is left to thediscretion of the practitioner, and will depend in part upon the site ofthe medical condition (such as the site of cancer). In a more preferredembodiment, the present combinations, compositions and kits of thepresent invention are administered intravenously.

The liquid combinations, compositions and kits of the invention, whetherthey are solutions, suspensions or other like form, can also include oneor more of the following: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordigylcerides, polyethylene glycols, glycerin, or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben; andagents for the adjustment of tonicity such as sodium chloride ordextrose. A parenteral combination or composition can be enclosed in anampoule, a disposable syringe or a multiple-dose vial made of glass,plastic or other material. Physiological saline is a preferred adjuvant.

For administration (e.g. intravenous) the combination, composition orkit may typically comprise the compound of formula I or a salt thereofat a dosage range of from 10 to 100 mg/kg body weight patient, theproteasome inhibitor at a dosage range of from 0.01 to 0.3 mg/kg bodyweight patient and the glucocorticoid at a dosage range of from 0.03 to1 mg/kg body weight patient. More preferably, the combination,composition or kit may typically comprise the compound of formula I or asalt thereof at a dosage range of from 40 to 80 mg/kg body weightpatient, the proteasome inhibitor at a dosage range of from 0.05 to 0.15mg/kg body weight patient and the glucocorticoid at a dosage range offrom 0.3 to 0.5 mg/kg body weight patient.

The combinations of the inventions may be formulated such that theproteasome inhibitor, the compound of formula I or a pharmaceuticallyacceptable salt thereof and, if present, the optional glucocorticoid ofthe combination are adapted for administration concurrently,sequentially or separately. Preferably, they are administeredconcurrently.

The combination, composition or kit of the present invention can beadministered by any convenient route, for example by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings.

In specific embodiments, it can be desirable to administer one or morecombinations, compositions or kits of the present invention orcombinations, compositions or kits locally to the area in need oftreatment. In one embodiment, administration can be by direct injectionat the site (or former site) of a cancer, tumor or neoplastic orpre-neoplastic tissue.

Pulmonary administration can also be employed, e.g. by use of an inhaleror nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant. Incertain embodiments, the combination, composition or kit of the presentinvention or compositions can be formulated as a suppository, withtraditional binders and carriers such as triglycerides.

The present combination, composition or kit can take the form ofsolutions, suspensions, emulsion, tablets, pills, pellets, capsules,capsules containing liquids, powders, sustained-release formulations,suppositories, emulsions, aerosols, sprays, suspensions, or any otherform suitable for use. Other examples of suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin.

The pharmaceutical combinations, compositions and kits can be preparedusing methodology well known in the pharmaceutical art. For example, acomposition intended to be administered by injection can be prepared bycombining the components of a kit of the present invention with water soas to form a solution. A surfactant can be added to facilitate theformation of a homogeneous solution or suspension.

The combinations, compositions and kits of the present invention areparticularly effective in the treatment of cancer.

The combinations of the present invention have been shown to haveexcellent activity against a wide variety of tumor cell types both invitro and in vivo, making them particularly interesting for developmentfor use in the treatment of cancer, e.g. hematologic cancer and breastcancer.

It has also discovered in the present work that the compound of formulaI or a salt thereof can be administered in combination with radiotherapyin the treatment of glioblastoma. Both in vitro and in vivo studiesshowed that a combination of the compound of formula I or a salt thereoftogether with radiotherapy was far more effective than radiotherapyalone. There is a prior disclosure in WO 2013/113838 of data for thecompound of formula I tested in the CNS Cancer (Glioma) cell linesSF-268, SF-295, SF-539, SNB-19, SNB-75 and U-251. These suggest activityfor the compound of formula I against glioblastoma when used on its own.

EXAMPLES

In the following examples, the compound having the following formula Iis referred to as EDO-S101 (or EDO in the Figures):

Example 1 EDO-S101 Combinations In Vitro—Multiple Myeloma MM1S Cell Line

EDO-S101 was combined in vitro with bortezomib and dexamethasone in themultiple myeloma MM1S cell line kindly provided by Steven Rosen atNorthwestern University, Chicago, Ill., USA. Activity was measured bythe MTT assay that is based on the metabolic bromide reduction from3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole (MTT), which isproduced by the mitochondrial enzyme succinate-dehydrogenase, turned toa blue-colored compound named formazan. The mitochondrial functionalityof the treated cells is then determined. This method has beenextensively used to measure cell proliferation and survival capacities.The remaining living cells are proportional to the amount of formazanproduced.

In brief the methodology was as follows:

-   -   30,000 MM1S cells per well were plated into the 96-well        microtiter plates.    -   EDO-S101 and PI dilutions were prepared in DMSO and        dexamethasone in ethanol and added into the wells to the final        concentrations indicated in the experiment.    -   Plates were incubated for 24-48-72 hours in the incubator at        37° C. in a humidified atmosphere in the presence of 5% CO₂/95%        air.    -   After the incubation 10 μL of MTT solution were added in each        well and incubated for 2 hours to allow formazan crystal        formation.    -   100 μl of a mix solution with SDS plus HCl (10 μL of HCl for        each 12 mL of SDS) was added to dissolve the formazan crystals.    -   Absorbance was read at 570 nm OD and use a reference wavelength        of 650 nm.    -   Cell viability (percentage) was obtained as follows: %        Viability=OD treated cells×100/OD control cells.    -   Each dose was tested in quadruplicate and each experiment was        performed at least twice.

The concentrations for the different drugs were ratio constant for allthe experiments. EDO-S101 at 500 nM, 1 μM, 2.5 μM; dexamethasone at 2.5nM; 5 nM; 10 nM; and bortezomib at 0.75 nM, 1.5 nM, 3 nM.

The results are as shown in Table 1 below and FIG. 1.

TABLE 1 48 H CI For experimental values Dexa 48 h EDO 48 h (nM) (nM) FaCI 2.5 500 0.43453 0.851 5 1000 0.56838 0.761 10 2000 0.683802 0.765 CIFor experimental values Bortz 48 h EDO 48 h (nM) (nM) Fa CI 0.75 5000.247333 1.087 1.5 1000 0.452958 1.230 3 2000 0.918526 0.627 CI Forexperimental values DOBLE Dexa 48 h Bortz 48 h (nM) (nM) Fa CI 2.5 0.750.413191 1.105 5 1.5 0.620757 0.879 10 3 0.935984 0.494 CI Forexperimental values Dexa 48 h Bortz 48 h EDO 48 h (nM) (nM) (nM) Fa CI2.5 0.75 150 0.455868 0.958 5 1.5 300 0.673133 0.789 10 3 600 0.9621730.404

The potency of the combination was quantitated with the Calcusynsoftware (biosoft, Ferguson, Mo., USA), which is based on the Chou Talaymethod (Chou et al., Adv. Enzyme Regul., 22, 27-55 (1984)), thatcalculates a combination index (CI) with the following interpretation:

-   -   CI 1>1: antagonist effect, CI=1: additive effect and CI<1        synergistic effect.

It can be seen from the FIG. 1 and from above that EDO-S101 showssynergy with both bortezomib and also shows synergy in a triplecombination with bortezomib and dexamethasone.

In a further experiment, the same constant dose of these drugs wasincubated for 72 hours instead of 48 hours. The results are as shown inTable 2 below and FIG. 2

TABLE 2 72 H CI For experimental values DEXA EDO (nM) (nM) Fa CI 2.5 5000.576413 0.682 5 1000 0.69365 0.836 10 2000 0.828332 0.829 CI Forexperimental values BORTZ EDO (nM) (nM) Fa CI 0.75 500 0.310537 1.3361.5 1000 0.780181 1.166 3 2000 0.999302 0.489 CI For experimental valuesDEXA BORTZ (nM) (nM) Fa CI 2.5 0.75 0.411026 1.441 5 1.5 0.865318 0.87610 3 1 0.017 CI For experimental values DEXA BORTZ EDO (nM) (nM) (nM) FaCI 2.5 0.75 500 0.607118 1.115 5 1.5 1000 0.923936 0.845 10 3 2000 10.017

Again, it can be seen from FIG. 2 and the above results in Table 2 thatEDO-S101 shows synergy with bortezomib and also shows synergy in atriple combination with bortezomib and dexamethasone.

Example 2 EDO-S101 Combinations In Vivo Against a Xenograft ofSubcutaneous Plasmacytoma

CB17-SCID mice (obtained from The Jackson Laboratory, Bar Harbor, Me.)were subcutaneously inoculated into the right flank with 3×10⁶ multiplemyeloma MM1S cells kindly provided by Steven Rosen at NorthwesternUniversity, Chicago, Ill., USA in 100 μL RPMI 1640 medium and 100 μL ofMatrigel (BD Biosciences). When tumours became palpable, mice wererandomized to 8 groups of treatment with 5 mice in each one.

The groups were:

-   -   Control (group treated with vehicle alone)    -   Bortezomib 1 mg/kg twice weekly intraperitoneal for three weeks    -   Dexamethasone 0.5 mg twice weekly intravenously for three weeks    -   EDO-S101 intravenously at doses of 30 mg/kg once weekly for 3        doses,    -   Bortezomib plus dexamethasone    -   Bortezomib plus EDO-S101    -   EDO-S101 plus dexamethasone    -   Triple combination of EDO-S101 plus Bortezomib and dexamethasone

Caliper measurements of the tumor diameters were performed every day,and the tumor volume was estimated as the volume of an ellipse using thefollowing formula: V=4/3 π×(a/2)×(b/2)2, where “a” and “b” correspond tothe longest and shortest diameter, respectively.

The tumour growth results are as shown in FIG. 3 in a plot of tumourgrowth (mm³) against the number of days of study. It can be seen thatthe combination of EDO-S101 and bortezomib results in tumour volumeslower than that seen with either agent alone while the triplecombination of EDO-S101, bortezomib and dexamethasone shows verysignificantly lower tumour volumes by the end of the study than any ofthe active agents individually.

Example 3 EDO-S101 Combinations In Vitro—Multiple Myeloma RPMI 8226 CellLine

Using the same test procedure as described in Example 1, but using themultiple myeloma RPMI 8226 cell line (obtained from DMSZ) in place ofthe MM1S cell line, combinations of EDO-S101 with bortezomib,carfilzomib and LU-102 were tested for activity in turn. Theconcentrations for the different drugs were ratio constant for all theexperiments. EDO-S101 at a concentration of 0, 2, 4, 8 μM; each ofbortezomib and carfilzomib at a concentration of 0, 5, 10, 20 nM; andLU-102 at a concentration of 0, 1, 3.3, 10 μM. Controls with bendamustinwere also performed.

The cell viability as a percentage of the untreated control was measuredand the results are as shown in FIG. 4. The figure shows clear synergyfor each of the three combinations with EDO-S101 in vitro againstmultiple myeloma RPMI 8226. The CI at 4 μM EDO-S101 and 20 nmcarfilzomib was 0.019 and the CI at 4 μM EDO-S101 and 3 μM LU-102 was0.109.

Example 4 EDO-S101 Combinations In Vitro—Multiple Myeloma Cell Line2013-10-16 MTS AMO Abzb

Using the same test procedure as described in Example 1, but using thebortezomib resistant multiple myeloma 2013-10-16 MTS AMO abzb cell line(generated at the Department of Oncology and Hematology of theKantonsspital St. Gallen by Prof. Dr. med. C. Driessen) in place of theMM1S cell line, combinations of EDO-S101 with bortezomib, carfilzomiband LU-102 were tested for activity in turn. The concentrations for thedifferent drugs were ratio constant and were 0, 2, 4, 8 μM for EDO-S101;0, 1.25, 2.5, 5, 10, 20 nM for each of bortezomib and carfilzomib; and0, 1, 3.3, 10 for LU-102.

The cell viability as a percentage of the untreated control was measuredand the results are as shown in FIG. 5. The figure shows clear synergyfor the combinations of carflizomib and LU-102 with EDO-S101 in vitroagainst the bortezomib resistant multiple myeloma 2013-10-16 MTS AMOabzb. The CI for the combinations of EDO-S101 and carfilzomib againstthis cell line was 0.11 and that for EDO-S101 and LU-102 was 0.25.

Example 5 EDO-S101 Combinations In Vitro—Mantle Cell Lymphoma Cell Line2014-01-15 MTS Jeko

Using the same test procedure as described in Example 1, ut using themantle cell lymphoma cell line 2014-01-15 MTS Jeko (obtained from LGCStandards S.a.r.l., 6, rue Alfred Kastler, BP 83076, F-67123 MolsheimCedex, France) in place of the MM1S cell line, combinations of EDO-S101with bortezomib, carfilzomib and LU-102 were tested for activity inturn. The concentrations for the different drugs were ratio constant forall the experiments and the same as in Example 3.

The cell viability as a percentage of the untreated control was measuredand the results are as shown in FIG. 6. The figure shows clear synergyfor each of the three combinations with EDO-S101 in vitro against mantlecell lymphoma cell line 2014-01-15 MTS Jeko. The CI at 2 μM EDO-S101 and20 nm bortezomib was 0.292; the CI at 2 μM EDO-S101 and 20 nmcarfilzomib was 0.206; and the CI at 2 μM EDO-S101 and 10 μM LU-102 was0.204.

Example 6 EDO-S101 Combinations In Vitro—Mantle Cell Lymphoma Cell Line2014-01-15 MTS Granta

Using the same test procedure as described in Example 1, but using themantle cell lymphoma cell line 2014-01-15 MTS Granta (obtained from LGCStandards S.a.r.l., 6, rue Alfred Kastler, BP 83076, F-67123 MolsheimCedex, France) in place of the MM1S cell line, combinations of EDO-S101with bortezomib, carfilzomib and LU-102 were tested for activity inturn. The concentrations for the different drugs were ratio constant forall the experiments and the same as in Example 3.

The cell viability as a percentage of the untreated control was measuredand the results are as shown in FIG. 7. The figure shows clear synergyfor each of the three combinations with EDO-S101 in vitro against mantlecell lymphoma cell line 2014-01-15 MTS Granta. The CI at 0.5 μM EDO-S101and 8 nm bortezomib was 0.025; the CI at 0.5 μM EDO-S101 and 8 nmcarfilzomib was 0.089; and the CI at 1 μM EDO-S101 and 3 μM LU-102 was0.078.

Example 7 EDO-S101 Combinations In Vitro—Basal Like Breast Cancer CellLine MTS MDA-MB468

Using the same test procedure as described in Example 1, but using thebasal like breast cancer cell line MTS MDA-MB468 (obtained from LGCStandards S.a.r.l., 6, rue Alfred Kastler, BP 83076, F-67123 MolsheimCedex, France) in place of the MM1S cell line, combinations of EDO-S101with bortezomib, carfilzomib and LU-102 were tested for activity inturn. The concentrations for the different drugs were ratio constant forall the experiments and were 0, 2, 4, 8 and 16 μM for EDO-S101; 0, 8, 16and 32 nM for each of bortezomib and carfilzomib; and 0, 1, 3.3 and 10μM for LU-102.

The cell viability as a percentage of the untreated control was measuredand the results are as shown in FIG. 8. The figure shows clear synergyfor each of the three combinations with EDO-S101 in vitro against thistriple negative breast cancer cell line MTS MDA-MB468.

Example 8 EDO-S101 Combinations In Vitro—Promyelocytic Leukemia CellLine HL-60

Using the same test procedure as described in Example 1, but using thepromyelocytic leukemia cell line HL-60 (obtained from LGC StandardsS.a.r.l., 6, rue Alfred Kastler, BP 83076, F-67123 Molsheim Cedex,France) in place of the MM1S cell line, combinations of EDO-S101 withbortezomib, carfilzomib and LU-102 were tested for activity in turn. Theconcentrations for the different drugs were ratio constant for all theexperiments and were 0, 1, 2 and 4 μM for EDO-S101; 0, 5, 10, 20 nM forbortezomib and carfilzomib; and LU-102 for 0, 1, 3.3, 10 μM.

The cell viability as a percentage of the untreated control was measuredand the results are as shown in FIG. 9. The figure shows clear synergyfor each of the three combinations with EDO-S101 in vitro againstpromyelocytic leukemia cell line HL-60. The CI at 1 μM EDO-S101 and 20nm bortezomibzomib was 0.051; the CI at 1 μM EDO-S101 and 20 nmcarfilzomib was 0.073; and the CI at 1 μM EDO-S101 and 3 μM LU-102 was0.387.

Example 9 EDO-S101 Combinations In Vitro—Acute Myeloid Leukemia CellLine U937

Using the same test procedure as described in Example 1, but using theacute myeloid leukemia cell line U937 (obtained from LGC StandardsS.a.r.l., 6, rue Alfred Kastler, BP 83076, F-67123 Molsheim Cedex,France) in place of the MM1S cell line, combinations of EDO-S101 withbortezomib, carfilzomib and LU-102 were tested for activity in turn. Theconcentrations for the different drugs were ratio constant for all theexperiments and were the same as in Example 8.

The cell viability as a percentage of the untreated control was measuredand the results are as shown in FIG. 10. The figure shows clear synergyfor each of the three combinations with EDO-S101 in vitro against basallike acute myeloid leukemia cell line U937. The CI at 2 μM EDO-S101 and10 nm bortezomib was 0.285; the CI at 2 μM EDO-S101 and 10 nmcarfilzomib was 0.272; and the CI at 2 μM EDO-S101 and 3 μM LU-102 was0.095.

Example 10 EDO-S101 Combinations In Vitro—B Cell Lymphoma Cell Line BJAB

Using the same test procedure as described in Example 1, but using the Bcell lymphoma cell line BJAB (germinal center line) (obtained from LGCStandards S.a.r.l., 6, rue Alfred Kastler, BP 83076, F-67123 MolsheimCedex, France) in place of the MM1S cell line, combinations of EDO-S101with bortezomib, carfilzomib and LU-102 were tested for activity inturn. The concentrations for the different drugs were ratio constant forall the experiments and were the same as in Example 8.

The cell viability as a percentage of the untreated control was measuredand the results are as shown in FIG. 11. The figure shows strong synergyfor the combination of EDO-S101 and carfilzomib in particular in vitroagainst B cell lymphoma cell line BJAB (germinal center line), while thecombination of EDO-S101 and bortezomib also showed synergy. The CI forthe combination of EDO-S101 and carfilzomib was 0.09, while the CI forthe combination of EDO-S101 and bortezomib was 0.62.

Example 11 EDO-S101 Combinations In Vitro—B Cell Lymphoma Cell LineOciLy3

Using the same test procedure as described in Example 1, but using the Bcell lymphoma cell line OciLy3 (ABC-type) (obtained from LGC StandardsS.a.r.l., 6, rue Alfred Kastler, BP 83076, F-67123 Molsheim Cedex,France) in place of the MM1S cell line, combinations of EDO-S101 withbortezomib, carfilzomib and LU-102 were tested for activity in turn. Theconcentrations for the different drugs were ratio constant for all theexperiments and were 0, 0.5, 1 and 2 μM for EDO-S101, 0, 5, 10 and 20 nMfor bortezomib and carfilzomib and 0, 1, 3.3 and 10 μM for LU-102.

The cell viability as a percentage of the untreated control was measuredand the results are as shown in FIG. 12. The figure shows strong synergyfor the combination of EDO-S101 and bortezomib in particular in vitroagainst B cell lymphoma cell line OciLy3 (ABC-type), while thecombination of EDO-S101 and carfilzomib also showed synergy. The CI forthe combination of EDO-S101 and carfilzomib was 0.59, while the CI forthe combination of EDO-S101 and bortezomib was 0.21.

Example 12 EDO-S101 Combinations In Vitro—B Cell Lymphoma Cell Line TMD8

Using the same test procedure as described in Example 1, but using the Bcell lymphoma cell line TMD8 (ABC-type) (obtained from LGC StandardsS.a.r.l., 6, rue Alfred Kastler, BP 83076, F-67123 Molsheim Cedex,France) in place of the MM1S cell line, combinations of EDO-S101 withbortezomib, carfilzomib and LU-102 were tested for activity in turn. Theconcentrations for the different drugs were ratio constant for all theexperiments and were the same as in Example 11.

The cell viability as a percentage of the untreated control was measuredand the results are as shown in FIG. 13. The figure shows strong synergyfor all combinations of EDO-S101 and proteasome inhibitor tested. The CIfor the combination of EDO-S101 and carfilzomib was 0.17, the CI for thecombination of EDO-S101 and bortezomib was 0.14 and the CI for thecombination of EDO-S101 and LU-102 was 0.63.

Example 13 EDO-S101 Combinations In Vitro—Triple Negative Breast CancerCell Line BT-549

Using the same test procedure as described in Example 1, but using thetriple negative breast cancer cell line BT-549 (obtained from LGCStandards S.a.r.l., 6, rue Alfred Kastler, BP 83076, F-67123 MolsheimCedex, France) in place of the MM1S cell line, combinations of EDO-S101with bortezomib, carfilzomib and LU-102 were tested for activity inturn. The concentrations for the different drugs were ratio constant forall the experiments and were 0, 1, 2 and 4 μM for EDO-S101; 0, 5, 10 and20 nM for each of bortezomib and carfilzomib; and 0, 1, 3.3 and 10 μMfor LU-102.

The cell viability as a percentage of the untreated control was measuredand the results are as shown in FIG. 14. The figure shows clear synergyfor each of the three combinations with EDO-S101 in vitro against triplenegative breast cancer cell line BT-549. The CI for the combination ofEDO-S101 and bortezomib was 0.14, the CI for the combination of EDO-S101and carfilzomib was 0.05 and the CI for the combination of EDO-S101 andLU-102 was 0.38.

Example 14 Combinations of Radiotherapy and EDO-S101 AgainstGlioblastoma Cell Lines In Vitro

For the U251MG glioblastoma cell line, the IC₅₀ was measured to be 6.60μM for EDO-S101 (compared to 30 μM for bendamustin and 20 fortemozolamide).

For the U87G glioblastoma cell line, the IC₅₀ was measured to be 1.36 μMfor EDO-S101 (compared to 50 μM for bendamustin and 20 fortemozolamide).

For the T98G glioblastoma cell line, the IC₅₀ was measured to be 7.70 μMfor EDO-S101 (compared to 52 μM for bendamustin and >100 fortemozolamide).

As can be seen from FIG. 15, the % survival rate for the glioblstomacells was considerably reduced when radiotherapy was used in combinationwith a dose of EDO-S101 (5 μM or 10 μM) compared to radiotherapy alone.

Example 15 Combinations of Radiotherapy and EDO-S101 AgainstGlioblastoma Cell Lines In Vivo U87MG, U251MG and T98G SubcutaneouslyInoculated Xenografts Treatments and Doses

-   -   Vehicle (control)    -   Radiotherapy (2Gy/5 consecutive days)    -   Temozolamide (16 mg/Kg for 5 consecutive days, po)    -   Temozolamide+radiotherapy    -   EDO-S101 (60 mg/Kg at day 1, 8 and 15 every 28 days, iv)    -   EDO-S101+radiotherapy

It was found that the time to progression of the tumours was increasedfrom approximately 17-18 days for the control for the U251MG mousexenograft model, to 42 days with a combination of radiotherapy andtemozolamide to over 50 days for EDO-S101 alone (significance P=0.924)to significantly over 50 days for a combination of EDO-S101 andradiotherapy (significance P=0.0359).

It was found that the time to progression of the tumours was increasedfrom approximately 15 days for the control for the U87MG mouse xenograftmodel, to 35 days with a combination of radiotherapy and temozolamide to40 days for EDO-S101 alone (significance P=2372) to significantly over50 days for a combination of EDO-S101 and radiotherapy (significanceP=0.0001).

Example 16 Activity of EDO-S101 Against Relapsed/Refractory MultipleMyeloma Models

A genetic rearrangement of the MYC locus, resulting in dysregulatedexpression of MYC, is the most common mutation in human multiplemyeloma. The genetically engineered Vk*MYC mouse model is based ondysregulation of MYC, and has been extensively validated as a clinicallyand biologically faithful model of untreated multiple myeloma. Ninedrugs or classes of drugs (DNA alkylators, glucocorticoids, proteasomeinhibitors, IMiDs, nab-paclitaxel, histone deacetylase inhibitors,TACI-Ig, perifosine and SNS-032, a CDK2,7,9 inhibitor) have beenpreviously reported with more than a 20% partial response rate in Vk*MYCMM. Among those, the first five also have greater than 20% PR inpatients with multiple myeloma for a positive predictive value of 56%.

EDO-S101 induced a high rate of response in Vk*MYC multiple myeloma thatwas sustained for more than three months in mice receiving only twodoses, one week apart. Remarkably EDO-S101 is the only drug that wasidentified with single agent activity in the very aggressive, multi-drugresistant Vk12653 transplant model of relapsed/refractory multiplemyeloma.

In conclusion, it can be seen that the compound of formula I (EDO-S101)show excellent activity in combination with proteasome inhibitors inacting both in vitro and in vivo against a wide range of myeloma,lymphoma, leukemia and breast cell lines. Furthermore, it can be seenthat the activity of many of these combinations is surprisinglysynergistic, and in many cases to a very significant degree. Yetfurther, it is seen in Examples 1 and 2 that triple combinationscomprising the compound of formula I, a proteasome inhibitor and aglucocorticoid such as dexamethasone showed particularly strong synergy.

As a result, it is to be expected that combinations of the compound offormula I of the present invention with a proteasome inhibitor,optionally comprising a glucocorticoid, will be of use in the treatmentof cancer, particularly hematologic cancers and breast cancer.

1. A combination comprising a proteasome inhibitor and a compound offormula I or a pharmaceutically acceptable salt thereof:


2. The combination according to claim 1, wherein the pharmaceuticallyacceptable salt of the compound of formula I is a hydrochloride,hydrobromide, hydroiodide, sulfate, bisulfate, sulfamate, nitrate,phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate,glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate,tosylate, salicylate, lactate, naphthalenesulfonate or acetate salt. 3.The combination according to claim 1, wherein the proteasome inhibitoris selected from the group consisting of bortezomib, carfilzomib,marizomib, delanzomib (CEP-18770), oprozomib (ONX 0912), ixazomib(MLN-9708), LU-102, and a pharmaceutically acceptable salt thereof. 4.The combination according to claim 1, wherein the proteasome inhibitoris selected from bortezomib, carfilzomib and LU-102.
 5. The combinationaccording to claim 1, further comprising a glucocorticoid.
 6. Thecombination according to claim 1, further comprising a glucocorticoidselected from the group consisting of dexamethasone, fluocinoloneacetonide and prednisone.
 7. The combination according to claim 1,further comprising the glucocorticoid dexamethasone.
 8. The combinationaccording to claim 1, further comprising one or more additionalpharmaceutically active agents.
 9. The combination according to claim 1,wherein the proteasome inhibitor, the compound of formula I orpharmaceutically acceptable salt thereof and, an optionalglucocorticoid, are adapted for administration concurrently,sequentially or separately.
 10. The combination according to claim 1,wherein the proteasome inhibitor, the compound of formula I orpharmaceutically acceptable salt thereof and, an optionalglucocorticoid, are adapted for administration concurrently.
 11. Thecombination according to claim 1, wherein the molar ratio of theproteasome inhibitor to the compound of formula I or pharmaceuticallyacceptable salt thereof in said combination is from 1:1000 to 1000:1.12. The combination according to claim 1, wherein the molar ratio of theproteasome inhibitor to the compound of formula I or pharmaceuticallyacceptable salt thereof in said combination is from 1:1000 to 10:1. 13.The combination according to claim 1, wherein the molar ratio of theproteasome inhibitor to the compound of formula I or pharmaceuticallyacceptable salt thereof in said combination is from 1:800 to 1:200. 14.The combination according to claim 1, wherein the molar ratio of theproteasome inhibitor to the compound of formula I or pharmaceuticallyacceptable salt thereof in said combination is from 1:700 to 1:400. 15.The combination according to claim 1, wherein the molar ratio of theproteasome inhibitor to the compound of formula I or pharmaceuticallyacceptable salt thereof in said combination is from 1:3 to 1:0.5. 16.The combination according to claim 1, comprising the compound of formulaI or the acetate salt thereof, and wherein the proteasome inhibitor isselected from bortezomib and carfilzomib, wherein the molar ratio of theproteasome inhibitor selected from bortezomib and carfilzomib to thecompound of formula I or pharmaceutically acceptable salt thereof insaid combination is from 1:700 to 1:400.
 17. The combination accordingto claim 1, comprising the compound of formula I or the acetate saltthereof and the proteasome inhibitor LU-102, wherein the molar ratio ofLU-102 to the compound of formula I or pharmaceutically acceptable saltthereof in said combination is from 1:3 to 1:0.5.
 18. The combinationaccording to claim 1, comprising the proteasome inhibitor and thecompound of formula I or pharmaceutically acceptable salt thereof,wherein the proteasome inhibitor and the compound of formula I or thepharmaceutically acceptable salt thereof form a synergistic combination.19. The combination according to claim 1, further comprising aglucocorticoid wherein the molar ratio of the proteasome inhibitor tothe compound of formula I or pharmaceutically acceptable salt thereof tothe glucocorticoid in said combination is from 1:1000:10 to 1000:1:20.20. The combination according to claim 1, further comprising aglucocorticoid wherein the molar ratio of the proteasome inhibitor tothe compound of formula I or pharmaceutically acceptable salt thereof tothe glucocorticoid used in said combination is from 1:1000:10 to1:100:2.
 21. The combination according to claim 1, further comprising aglucocorticoid wherein the molar ratio of the proteasome inhibitor tothe compound of formula I or pharmaceutically acceptable salt thereof tothe glucocorticoid used in said combination is from 1:700:4 to 1:400:3.22. The combination according to claim 1, comprising a proteasomeinhibitor selected from bortezomib and carfilzomib, the compound offormula I or the acetate salt thereof and dexamethasone, wherein themolar ratio of the proteasome inhibitor selected from bortezomib andcarfilzomib to the compound of formula I or the acetate salt thereof todexamethasone in said combination is from 1:700:4 to 1:400:3.
 23. Thecombination according to claim 1, comprising the proteasome inhibitorLU-102, the compound of formula I or the acetate salt thereof anddexamethasone, wherein the molar ratio of LU-102 to the compound offormula I or the acetate salt thereof to dexamethasone in saidcombination is from 1:3:4 to 1:0.5:3.
 24. The combination according toclaim 1, comprising the proteasome inhibitor, the compound of formula Ior pharmaceutically acceptable salt thereof and a glucocorticoid,wherein the proteasome inhibitor, the compound of formula I or thepharmaceutically acceptable salt thereof and the glucocorticoid form asynergistic combination.
 25. A pharmaceutical composition comprising apharmaceutically acceptable diluent or carrier and a combinationaccording to claim
 1. 26. A kit comprising a combination according toclaim 1, and optionally, instructions for treating a patient. 27-42.(canceled)
 43. A method of treating cancer in a patient in need thereofcomprising administering to said patient a combination according toclaim
 1. 44. The method according to claim 43, wherein said cancer isselected from a hematologic cancer and breast cancer.
 45. The methodaccording to claim 43, wherein said cancer is a hematologic cancerselected from multiple myeloma, lymphoma and leukemia.
 46. The methodaccording to claim 43, wherein said cancer is multiple myeloma selectedfrom active myeloma, plasmacytoma, light chain myeloma and non-secretorymyeloma.
 47. The method according to claim 43, wherein said cancer islymphoma selected from Hodgkin lymphoma and non-Hodgkin lymphoma. 48.The method according to claim 43, wherein said cancer is leukemiaselected from acute lymphoblastic leukemia (ALL), chronic lymphocyticleukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia(CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia(T-PLL), large granular lymphocytic leukemia and T-cell acutelymphoblastic leukemia.
 49. The method according to claim 43, whereinsaid cancer is breast cancer selected from claudin-low tumors,basal-like tumors, human epidermal growth factor receptor 2 (HER2)positive tumors, luminal A tumors and luminal B tumors.
 50. The methodaccording to claim 43, wherein said cancer is a triple-negative breastcancer.
 51. The method according to claim 43, wherein in said method theproteasome inhibitor, the compound of formula I or pharmaceuticallyacceptable salt thereof and, an optional glucocorticoid, areadministered concurrently, sequentially or separately.
 52. The methodaccording to claim 43, wherein in said method the proteasome inhibitor,the compound of formula I or pharmaceutically acceptable salt thereofand, an optional glucocorticoid, are administered concurrently.
 53. Themethod according to claim 43, wherein the compound of formula I orpharmaceutically acceptable salt thereof is administered to the patientin need thereof at a dosage range of 10 to 100 mg/kg body weightpatient.
 54. The method according to claim 43, wherein the compound offormula I or pharmaceutically acceptable salt thereof is administered tothe patient in need thereof at a dosage range of 40 to 80 mg/kg bodyweight patient.
 55. The method according to claim 43, wherein theproteasome inhibitor is administered to the patient at a dosage range of0.01 to 0.3 mg/kg body weight patient.
 56. The method according to claim43, wherein the proteasome inhibitor is administered to the patient at adosage range of 0.05 to 0.15 mg/kg body weight patient.
 57. The methodaccording to claim 43, wherein the combination further comprises aglucocorticoid, and wherein in the method, the glucocorticoid isadministered at a dosage range of from 0.1 to 1.0 mg/kg body weightpatient.
 58. The method according to claim 43, wherein the combinationfurther comprises a glucocorticoid, and wherein in the method, theglucocorticoid is administered at a dosage range of from 0.3 to 0.5mg/kg body weight patient.